Complete genome sequences of Cytobacillus sp., Domibacillus sp., Enterobacter sp., Neisseria sp., Pseudomonas sp., and Streptococcus sp. strains from human clinical infections collected at diagnostic units in 2020.

Members of Bacillota and Pseudomonadota phyla are frequently considered bacterial infectious agents in humans. As part of a large sequencing project of clinically relevant pathogens, we hybrid-assembled complete genomes of Cytobacillus, Domibacillus, Enterobacter, Neisseria, Pseudomonas, and Streptococcus species isolated from clinical specimens.

Whole genomes from bacteria collected at diagnostic units around the world 2020.

The Two Weeks in the World research project has resulted in a dataset of 3087 clinically relevant bacterial genomes with pertaining metadata, collected from 59 diagnostic units in 35 countries around the world during 2020. A relational database is available with metadata and summary data from selected bioinformatic analysis, such as species prediction and identification of acquired resistance genes.

Reduced Birth Weight Caused by Sextuple Drug-Resistant Plasmodium falciparum Infection in Early Second Trimester.

Mutations in the Plasmodium falciparum genes Pfdhfr and Pfdhps, particularly the sextuple mutant haplotype threatens the antimalarial effectiveness of sulfadoxine-pyrimethamine (SP) as intermittent preventive treatment during pregnancy (IPTp). To explore the impact of sextuple mutant haplotype infections on outcome measures after provision of IPTp with SP, we monitored birth outcomes in women followed up from before conception or from the first trimester until delivery. Women infected with sextuple haplotypes, in the early second trimester specifically, delivered newborns with a lower birth weight compared with women who did not have malaria during pregnancy (difference, -267 g; 95% confidence interval, -454 to -59; P = .01) and women infected with less SP-resistant haplotypes (-461 g; -877 to -44; P = .03). Thus, sextuple haplotype infections seem to affect the effectiveness of SP for IPTp and directly affect birth outcome by lowering birth weight. Close monitoring and targeted malaria control during early pregnancy is therefore crucial to improving birth outcomes.

Proof of concept: used malaria rapid diagnostic tests applied for parallel sequencing for surveillance of molecular markers of anti-malarial resistance in Bissau, Guinea-Bissau during 2014-2017.

BACKGROUND: Large-scale surveillance of molecular markers of anti-malarial drug resistance is an attractive method of resistance monitoring, to complement therapeutic efficacy studies in settings where the latter are logistically challenging. METHODS: Between 2014 and 2017, this study sampled malaria rapid diagnostic tests (RDTs), used in routine clinical care, from two health centres in Bissau, Guinea-Bissau. In order to obtain epidemiological insights, RDTs were collected together with patient data on age and sex. A subset of positive RDTs from one of the two sites (n = 2184) were tested for Plasmodium DNA content. Those testing positive for Plasmodium DNA by PCR (n = 1390) were used for library preparation, custom designed dual indexing and next generation Miseq targeted sequencing of Plasmodium falciparum genes pfcrt, pfmdr1, pfdhfr, pfdhps and pfk13. RESULTS: The study found a high frequency of the pfmdr1 codon 86N at 88-97%, a significant decrease of the pfcrt wildtype CVMNK haplotype and elevated levels of the pfdhfr/pfdhps quadruple mutant ranging from 33 to 51% between 2014 and 2017. No polymorphisms indicating artemisinin tolerance were discovered. The demographic data indicate a large proportion of young adults (66%, interquartile range 11-28 years) presenting with P. falciparum infections. While a total of 5532 gene fragments were successfully analysed on a single Illumina Miseq flow cell, PCR-positivity from the library preparation varied considerably from 13 to 87% for different amplicons. Furthermore, pre-screening of samples for Plasmodium DNA content proved necessary prior to library preparation. CONCLUSIONS: This study serves as a proof of concept for using leftover clinical material (used RDTs) for large-scale molecular surveillance, encompassing the inherent complications regarding to methodology and analysis when doing so. Factors such as RDT storage prior to DNA extraction and parasitaemia of the infection are likely to have an effect on whether or not parasite DNA can be successfully analysed, and are considered part of the reason the data yield is suboptimal. However, given the necessity of molecular surveillance of anti-malarial resistance in settings where poor infrastructure, poor economy, lack of educated staff and even surges of political instability remain major obstacles to performing clinical studies, obtaining the necessary data from used RDTs, despite suboptimal output, becomes a feasible, affordable and hence a justifiable method.

Molecular assays for antimalarial drug resistance surveillance: A target product profile.

Antimalarial drug resistance is a major constraint for malaria control and elimination efforts. Artemisinin-based combination therapy is now the mainstay for malaria treatment. However, delayed parasite clearance following treatment with artemisinin derivatives has now spread in the Greater Mekong Sub region and may emerge or spread to other malaria endemic regions. This spread is of great concern for malaria control programmes, as no alternatives to artemisinin-based combination therapies are expected to be available in the near future. There is a need to strengthen surveillance systems for early detection and response to the antimalarial drug resistance threat. Current surveillance is mainly done through therapeutic efficacy studies; however these studies are complex and both time- and resource-intensive. For multiple common antimalarials, parasite drug resistance has been correlated with specific genetic mutations, and the molecular markers associated with antimalarial drug resistance offer a simple and powerful tool to monitor the emergence and spread of resistant parasites. Different techniques to analyse molecular markers associated with antimalarial drug resistance are available, each with advantages and disadvantages. However, procedures are not adequately harmonized to facilitate comparisons between sites. Here we describe the target product profiles for tests to analyse molecular markers associated with antimalarial drug resistance, discuss how use of current techniques can be standardised, and identify the requirements for an ideal product that would allow malaria endemic countries to provide useful spatial and temporal information on the spread of resistance.

Direct whole-genome sequencing of Plasmodium falciparum specimens from dried erythrocyte spots.

BACKGROUND: Plasmodium falciparum malaria remains a major health burden and genomic research represents one of the necessary approaches for continued progress towards malaria control and elimination. Sample acquisition for this purpose is troublesome, with the majority of malaria-infected individuals living in rural areas, away from main infrastructure and the electrical grid. The aim of this study was to describe a low-tech procedure to sample P. falciparum specimens for direct whole genome sequencing (WGS), without use of electricity and cold-chain. METHODS: Venous blood samples were collected from malaria patients in Bandim, Guinea-Bissau and leukocyte-depleted using Plasmodipur filters, the enriched parasite sample was spotted on Whatman paper and dried. The samples were stored at ambient temperatures and subsequently used for DNA-extraction. Ratios of parasite:human content of the extracted DNA was assessed by qPCR, and five samples with varying parasitaemia, were sequenced. Sequencing data were used to analyse the sample content, as well as sample coverage and depth as compared to the 3d7 reference genome. RESULTS: qPCR revealed that 73% of the 199 samples were applicable for WGS, as defined by a minimum ratio of parasite:human DNA of 2:1. WGS revealed an even distribution of sequence data across the 3d7 reference genome, regardless of parasitaemia. The acquired read depths varied from 16 to 99×, and coverage varied from 87.5 to 98.9% of the 3d7 reference genome. SNP-analysis of six genes, for which amplicon sequencing has been performed previously, confirmed the reliability of the WGS-data. CONCLUSION: This study describes a simple filter paper based protocol for sampling P. falciparum from malaria patients for subsequent direct WGS, enabling acquisition of samples in remote settings with no access to electricity.

Country-Wide Surveillance of Molecular Markers of Antimalarial Drug Resistance in Senegal by Use of Positive Malaria Rapid Diagnostic Tests.

In Senegal, antimalarial drugs used in treatment and prevention of malaria are one of the main reasons for the current success in controlling malaria. However, the successful control of malaria is highly dependent on continued effectiveness of these drugs which may be compromised by the spread of drug resistance. Therefore, surveillance of drug resistance in the malaria parasites is essential. The objective of this pilot study was to test the feasibility of routinely sampled malaria rapid diagnostic tests (RDTs) at a national scale to assess the temporal changes in the molecular profiles of antimalarial drug resistance markers of Plasmodium falciparum parasites. Overall, 9,549 positive malaria RDTs were collected from 14 health facilities across the country. A limited random set of RDTs were analyzed regarding Pfcrt gene polymorphisms at codon 72-76. Overall, a high but varied prevalence (> 50%) of the wild-type CVMNK haplotype was observed including a higher CVMNK prevalence in the northern part (75%) compared with the southern part of the country (59%). With caution, the study provides a proof of concept that reuse of discarded P. falciparum positive RDTs can be applied in large-scale surveillance of antimalarial drug resistance.

High throughput resistance profiling of Plasmodium falciparum infections based on custom dual indexing and Illumina next generation sequencing-technology.

Genetic polymorphisms in P. falciparum can be used to indicate the parasite's susceptibility to antimalarial drugs as well as its geographical origin. Both of these factors are key to monitoring development and spread of antimalarial drug resistance. In this study, we combine multiplex PCR, custom designed dual indexing and Miseq sequencing for high throughput SNP-profiling of 457 malaria infections from Guinea-Bissau, at the cost of 10 USD per sample. By amplifying and sequencing 15 genetic fragments, we cover 20 resistance-conferring SNPs occurring in pfcrt, pfmdr1, pfdhfr, pfdhps, as well as the entire length of pfK13, and the mitochondrial barcode for parasite origin. SNPs of interest were sequenced with an average depth of 2,043 reads, and bases were called for the various SNP-positions with a p-value below 0.05, for 89.8-100% of samples. The SNP data indicates that artemisinin resistance-conferring SNPs in pfK13 are absent from the studied area of Guinea-Bissau, while the pfmdr1 86 N allele is found at a high prevalence. The mitochondrial barcodes are unanimous and accommodate a West African origin of the parasites. With this method, very reliable high throughput surveillance of antimalarial drug resistance becomes more affordable than ever before.

Different origin and dispersal of sulfadoxine-resistant Plasmodium falciparum haplotypes between Eastern Africa and Democratic Republic of Congo.

Sulfadoxine/pyrimethamine (SP) is still used for malaria control in sub-Saharan Africa; however, widespread resistance is a major concern. This study aimed to determine the dispersal and origin of sulfadoxine resistance lineages in the Democratic Republic of the Congo compared with East African Plasmodium falciparum dihydropteroate synthetase (Pfdhps) haplotypes. The analysis involved 264 isolates collected from patients with uncomplicated malaria from Tanzania, Uganda and DR Congo. Isolates were genotyped for Pfdhps mutations at codons 436, 437, 540, 581 and 613. Three microsatellite loci (0.8, 4.3 and 7.7 kb) flanking the Pfdhps gene were assayed. Evolutionary analysis revealed a shared origin of Pfdhps haplotypes in East Africa, with a distinct population clustering in DR Congo. Furthermore, in Tanzania there was an independent distinct origin of Pfdhps SGEGA resistant haplotype. In Uganda and Tanzania, gene flow patterns contribute to the dispersal and shared origin of parasites carrying double- and triple-mutant Pfdhps haplotypes associated with poor outcomes of intermittent preventive treatment during pregnancy using SP (IPTp-SP). However, the origins of the Pfdhps haplotypes in DR Congo and Eastern Africa sites are different. The genetic structure demonstrated a divergent and distinct population cluster predominated by single-mutant Pfdhps haplotypes at the DR Congo site. This reflects the limited dispersal of double- and triple-mutant Pfdhps haplotypes in DR Congo. This study highlights the current genetic structure and dispersal of high-grade Pfdhps resistant haplotypes, which is important to guide implementation of SP in malaria chemoprevention strategies in the region.

Molecular monitoring of Plasmodium falciparum super-resistance to sulfadoxine-pyrimethamine in Tanzania.

BACKGROUND: Sulfadoxine-pyrimethamine (SP) is recommended for prophylactic treatment of malaria in pregnancy while artemisinin combination therapy is the recommended first-line anti-malarial treatment. Selection of SP resistance is ongoing since SP is readily available in health facilities and in private drug shops in sub-Saharan Africa. This study reports on the prevalence and distribution of Pfdhps mutations A540E and A581G in Tanzania. When found together, these mutations confer high-level SP resistance (sometimes referred to as 'super-resistance'), which is associated with loss in protective efficacy of SP-IPTp. METHODS: DNA samples were extracted from malaria-positive blood samples on filter paper, used malaria rapid diagnostic test strips and whole blood collected from eight sites in seven administrative regions of Tanzania. PCR-RFLP and SSOP-ELISA techniques were used to genotype the A540E and A581G Pfdhps. Data were analysed using SPSS version 18 while Chi square and/or Fischer Exact tests were used to compare prevalence between regions. RESULTS: A high inter-regional variation of Pfdhps-540E was observed (χ(2) = 76.8, p < 0.001). High inter-regional variation of 581G was observed (FE = 85.3, p < 0.001). Both Tanga and Kagera were found to have the highest levels of SP resistance. A high prevalence of Pfdhps-581G was observed in Tanga (56.6 %) in northeastern Tanzania and in Kagera (20.4 %) in northwestern Tanzania and the 540-581 EG haplotype was found at 54.5 and 19.4 %, respectively. Pfdhps-581G was not detected in Pwani and Lindi regions located south of Tanga region. CONCLUSIONS: Selection of SP super-resistant Pfdhps A581G is highest in northern Tanzania. Variation in distribution of SP resistance is observed across the country: northeastern Tanga region and northwestern Kagera region have highest prevalence of SP super-resistance markers, while in Pwani and Lindi in the southeast the prevalence of super-resistance was zero. More studies should be conducted to understand the factors underlying the remarkable heterogeneity in SP resistance in the country.

Independent origin of plasmodium falciparum antifolate super-resistance, Uganda, Tanzania, and Ethiopia.

Super-resistant Plasmodium falciparum threatens the effectiveness of sulfadoxine-pyrimethamine in intermittent preventive treatment for malaria during pregnancy. It is characterized by the A581G Pfdhps mutation on a background of the double-mutant Pfdhps and the triple-mutant Pfdhfr. Using samples collected during 2004-2008, we investigated the evolutionary origin of the A581G mutation by characterizing microsatellite diversity flanking Pfdhps triple-mutant (437G+540E+581G) alleles from 3 locations in eastern Africa and comparing it with double-mutant (437G+540E) alleles from the same area. In Ethiopia, both alleles derived from 1 lineage that was distinct from those in Uganda and Tanzania. Uganda and Tanzania triple mutants derived from the previously characterized southeastern Africa double-mutant lineage. The A581G mutation has occurred multiple times on local Pfdhps double-mutant backgrounds; however, a novel microsatellite allele incorporated into the Tanzania lineage since 2004 illustrates the local expansion of emergent triple-mutant lineages.